The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans.

Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-γ, IL-10, and IL-1β levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RHΔrop16 and RHΔrop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-γ, IL-10, IL-1β, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RHΔrop16, whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1β was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-γ production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response.

Apicomplexa are an extremely diverse group of unicellular organisms that infect humans and other animals. Despite the great advances in combating infectious diseases over the past century, these parasites still have a tremendous social and economic burden on human societies, particularly in tropical and subtropical regions of the world. Proteases from apicomplexa have been characterized at the molecular and cellular levels, and central roles have been proposed for proteases in diverse processes. In this work, 16 new genes encoding for trypsin proteases are identified in 8 apicomplexan genomes by a genome-wide survey. Phylogenetic analysis suggests that these genes were gained through both intracellular gene transfer and vertical gene transfer. Identification, characterization and understanding of the evolutionary origin of protease-mediated processes are crucial to increase the knowledge and improve the strategies for the development of novel chemotherapeutic agents and vaccines.

BackgroundThe interactions between pathogen proteins and their hosts allow pathogens to manipulate host cellular mechanisms to their advantage. The identification of host proteins that are targeted by virulent pathogen proteins is crucial to increase our understanding of infection mechanisms and to propose new therapeutics that target pathogens. Understanding the virulence mechanisms of pathogens requires a detailed molecular description of the proteins involved, but acquiring this knowledge is time consuming and prohibitively expensive. Therefore, we develop a statistical method based on hypothesis testing to compare the time series obtained from conversion of the physicochemical characteristics of the amino acids that form the primary structure of proteins and thus to propose potential functional relation between proteins. We called this algorithm the multiple spectral comparison algorithm (MSCA); the MSCA was inspired by the BLASTP tool and was implemented in R code. The algorithm compares and relates multiple time series according to their spectral similarities, and the biological relation between them could be interpreted as either a similar function or protein-protein interaction (PPI).ResultsA simulation study showed that the MSCA works satisfactorily well when we compare unequal time series generated from ARMA processes because its power was close to 1. The MSCA presented a 70% average accuracy of detecting protein interactions using a threshold of 0.7 for our spectral measure, indicating that this algorithm could predict novel PPIs and pathogen-host interactions (PHIs) with acceptable confidence. The MSCA also was validated by its identification of well-known interactions of the human proteins MAGI1, SCRIB and JAK1, as well as interactions of the virulence proteins ROP16, ROP18, ROP17 and ROP5. We verified the spectral similarities for human intraspecific PPIs and PHIs that were previously demonstrated experimentally by other authors. We suggest that human GBP (GTPase group induced by interferon) and the CREB transcription factor family could be human substrates for the complex of ROP18, ROP17 and ROP5.ConclusionsUsing multiple-hypothesis testing between the spectral densities of a set of unequal time series, we developed an algorithm that is able to identify the similarities or interactions between a set of proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0599-8) contains supplementary material, which is available to authorized users.

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